Carcinus maenas is known for its high acclimation potential to varying environmental abiotic conditions. A high ability for ion and acid-base regulation is mainly based on an efficient regulation apparatus located in gill epithelia. However, at present it is neither known which ion transport proteins play a key role in the acid-base compensation response nor how gill epithelia respond to elevated seawater pCO2 as predicted for the future. In order to promote our understanding of the responses of green crab acid-base regulatory epithelia to high pCO2, Baltic Sea green crabs were exposed to a pCO2 of 400 Pa. Gills were screened for differentially expressed gene transcripts using a 4,462-feature microarray and quantitative real-time PCR.The green crab pCO2. However, 2% of all investigated transcripts were significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO2 stress. Most of the genes known to code for proteins involved in osmo- and acid-base regulation, as well as cellular stress response, were were not impacted by elevated pCO2. However, after one week of exposure, significant changes were detected in a calcium-activated chloride channel, a hyperpolarization activated nucleotide-gated potassium channel, a tetraspanin, and an integrin. Furthermore, a putative syntaxin-binding protein, a protein of the transmembrane 9 superfamily, and a Cl-/HCO3- exchanger of the SLC 4 family were differentially regulated. These genes were also affected in a previously published hypoosmotic acclimation response study.Crabs responded mainly through fine scale adjustment of gene expression to elevated C. maenas gill gene expression indicates that (1) seawater acidification does not act as a strong stressor on the cellular level in gill epithelia; (2) the response to hypercapnia is to some degree comparable to a hypoosmotic acclimation response; (3) the specialization of each of the posterior gill arches might go beyond what has been demonstrated up to date; and (4) a re-configuration of gill epithelia might occur in response to hypercapnia.The moderate, but specific response of  In teleost fish, cephalopods, and decapod crustaceans, the majority of the acid-base relevant ion regulatory apparatus is located in gill epithelia. It is thought that net proton extrusion is primarily achieved via active (V-type H+-ATPase) and secondarily active ion transport molecules , with a strong supporting role of carbonic anhydrases (CAs) and Na+/K+-ATPase  + [HCO3-] + [CO32-]) was measured according to Dickson and Millero ; table Seawater total alkalinity  for the long-term experiment were part of a study by Appelhans et al. (under review) that was conducted in parallel. Experimental animals for this long-term study were caught in traps in April 2009 in Kiel Fjord  at the IFM-GEOMAR pier (westshore building) in 3 to 4 m water depth . Animals with a carapace width of 4.5 ± 0.5 cm were chosen irrespectively of their sex . Crabs were exposed to control  and elevated pCO2 . Animals were fed with 2 mussels (size = 2.50 ± 0.35 cm) three times a week. To evaluate the carbonate system parameters, water samples of three aquaria per treatment level were taken at the beginning, the intermediate phase and at the end of the experiment, respectively. Analysis of CT was coulometric  and AT analyzed via potentiometric titration  using the RNeasy Midi-Kit from Qiagen (#75144). Gills were homogenized with an OMNI international TH/G7-195STW for 50 s in 4 mL buffer RLT from the Qiagen kit. Extraction then followed the manufacturer's description. RNA quality was monitored by electrophoresis via the Agilent 2100 Bioanalyzer system. Each RNA sample was then reversely transcribed into cDNA and in parallel fluorescence-labeled using the SuperScript™ Plus Direct cDNA Labeling System from Invitrogen (#L1015-06), following the provided protocol. Using both anchored oligo(dT)20 and random hexamer primers included in the kit, control cDNA was labeled with AlexaFluor555  while cDNA of the CO2-treatment samples was labeled with AlexaFluor647 .RNA from the first short term experiment (2009) was extracted from whole left gills #7 and #9 , was performed by Towle et al.  was also investigated in the long-term exposed animals  and did not show a significant change.Out of 137 identified transcripts associated with the cellular stress response (according to ), only 3DN202592.1]), also suggests structural modification of gill membranes. It was up-regulated in both gills on both sampling days (sign test with p < 0.05). This transcript was also identified to be up-regulated in the low salinity study [DV944030.1], included in the GO-class "cell") was found to be strongly down-regulated during short-term hypercapnia (1.3-fold) on day 3 and slightly during low salinity exposure. A putative Integrin-alpha-7 [GenBank:DY307809.1] was identified to be strongly down-regulated  on day 7 . Significant up-regulation of 13 genes  from this group resulted in the over-representation of this GO-term in gill 9 , a gene that was not altered in gill 9, regarding both, day 3 and 7. On the other hand, gene expression of a calcium-activated chloride channel [GenBank:DW584691.1] was only significantly and strongly affected in gill 9 on both days of the short-term exposure (sign test and F-test with p < 0.05). Additionally, according to the enrichment analysis, structural changes in gill epithelia are linked mainly to gill 9.Although no effect of the factor "gill" on individual gene expression was identified by variance-based linear modeling, Wilcoxon's matched pairs test identified a significant difference in the overall gene expression patterns in the different experimental blocks  resulted in smaller changes in expression levels in fewer transcripts: only 8 transcripts were regulated > 1.5-fold (log2-ratio > |0.60|) in the present study, while 533 transcripts were regulated > 1.5-fold in the low salinity acclimation study; maximum expression changes of up to 14-fold (log2-ratio = 3.8) were observed in the low salinity acclimation study , short-y Figure , 4. Neve2 between 15 to 79%, Figure DN739347.1]), the transcript was slightly, but significantly down-regulated in the microarray analysis, whereas it was up-regulated in the qRT-PCR analysis. Variation in both, qRT-PCR and in the microarray experiment was high.In the long-term experiment, no significant changes in expression were found, likely due to the high biological variance within samples . This transcript was also the most strongly up-regulated gene in gill 8 in response to low salinity transfer [DW250369.1], only identified by F-test, was another highly up-regulated transcript on day 3, while it showed strong down-regulation on day 7. In gill 9, the most strongly down-regulated gene was identified on day 7 as a 23S ribosomal RNA gene [GenBank:DN796131.1] with a median log2-ratio of -0.87 ± 0.46 . This gene was strongly up-regulated during the low-salinity experiment. Additionally, a different 23S ribosomal gene was found to be equally strongly affected on day 7 in gill 7 . The most strongly up-regulated transcript in gill 9 at day 3 was a calcium-activated chloride channel [GenBank:DW584691.1] with a median log2-ratio of +0.81 ± 0.34 . It was also the most highly up-regulated transcript in the long-term exposed animals , although not tested significant due to high variance between replicates. Additionally, rRNA intron-encoded homing endonuclease [GenBank:DV944193.1] was identified by F-test as being differentially expressed. Besides 4 unknown, but strongly changed transcripts . In addition, a hyperpolarization-activated cyclic nucleotide-gated potassium channel , was significantly down-regulated in both gills on day 7  with a tendency for down-regulation in both gills on day 3. In the low-salinity study, it was only slightly down-regulated , + 3.4-fold), the Na+/K+-ATPase alpha subunit , a member of the anion/bicarbonate transporter family (abts-3) , - 2.4-fold), the glycosyl-phosphatidylinositol-linked carbonic anhydrase VII  and the Cl-/HCO3- exchanger .None of the acid-base regulatory candidate genes were regulated more than 1.3-fold in our microarray experiment at days 3 and 7. Of 9 candidate genes tested on the microarray, only 2 were significantly regulated with respect to controls. Among them were a Clle et al  found 5 2 ranged from 15 to 79%). Only in the case of the sodium-hydrogen exchanger NHE3, variance was smaller (1.3%).In the long- term acclimation study (11 weeks), no significant changes in expression could be found, but variances were extremely high . The enrichment of HCO3- in the crabs' hemolymph may be mediated by altered ion regulation processes in the gills and thus can be expected to also leave a footprint in the gills' transcriptome. However, long-term hypercapnia incubations may additionally result in other physiological responses, pointing to trade-offs in energy allocation in favor of extra-/intracellular ion homeostasis vs. protein metabolism and growth, as has been concluded for molluscs, teleost fish and echinoderms . During hypercapnia acclimation, Deigweiher et al. ), and a vacuolar H+-ATPase [GenBank:DY656042.1] were not affected. Another important transporter for acid-base regulation, a Na+/H+ exchanger (NHE3) was not significantly regulated in C. maenas gill tissue in the long- term experiment. C. maenas also possesses two branchial isoforms of carbonic anhydrase (CA) in its posterior gills, a membrane-associated and a cytoplasmic form [2O and CO2 to HCO3- and H+ and vice versa. It is of great importance in osmoregulation , acid-bstaceans ,83. HoweC. maenas already works at a high load due to the demands of a hyposmotic habitat with large fluctuations in pCO2. Therefore, only moderate mRNA expression level changes might be necessary to compensate hypercapnia in C. maenas. Compensation of the ion regulatory apparatus might additionally take place on the post-transcriptional level or is facilitated by transcripts not included on this microarray. It has to be considered though, that to some extend, effects might be hidden under the experimental error. Nevertheless, those few genes for which we have identified relatively strong changes in expression levels are particularly interesting. They are likely key players of hypercapnia acclimation of crustacean gill tissues.We suggest that the ion regulatory apparatus of Baltic Carcinus maenas to elevated seawater pCO2 based on the gill transcriptome does not suggest that seawater acidification acts as a strong stressor for the western Baltic population of this species. Following short-term hypercapnia, the low but specific responses of the gills indicate that (1) the response to hypercapnia is partially similar to the response to hypoosmotic conditions; (2) a multispanning membrane protein (TM9SF4), a calcium-activated chloride channel (CaCC) and a hyperpolarization-activated cyclic nucleotide-gated potassium channel (HCN) are strongly regulated in response to hypercapnia and may be involved in acid-base regulation; (3) posterior gills might respond differently to hypercapnia; and (4) a re-configuration of the epithelial gill membrane, including the involvement of a tetraspanin-integrin complex, might occur.The response of Due to the low salinity environment in the Baltic Sea in this hypercapnia study, effects on distinct gene expression levels to one of the respective abiotic factors might be influenced by the other. It is therefore essential to study both abiotic factors separately and in more detail. Here, potential pathological effects of the long-term consequences of elevated blood bicarbonate concentrations on various tissues and organs should also deserve particular attention. We also expect that stronger effects of hypercapnia are to be found on the proteome and metabolome level.SF carried out the short-term incubations, microarray experiments and real-time PCR, analyzed the results and drafted the manuscript. Most of the facts displayed here are content of SF's Diploma thesis at the IFM-GEOMAR, Kiel, Germany 2010. RK contributed to the analysis, illustration and interpretation of the data and the preparation of the manuscript. YA carried out the long term incubation experiments and participated in the sampling procedure. DWT passed away during the preparation of the manuscript; before his untimely death, he significantly contributed to the microarray and study design and coordination, and provided the microarray facilities. FM and MZ conceived the study, participated in its design, coordination and analysis, supplied infrastructure and material and helped to draft the manuscript. All authors read and approved the final manuscript.Tables S1-S6. Carcinus maenas the microarray assay applied in this studyTable S1. Annotation details on transcripts for the . Annotation results on transcripts for the Carcinus maenas microarray assay applied in this study from a newly performed bioinformatic analysis conducted with the internet portal Blast2GO [Blast2GO  in 2009 Blast2GO . IncludeCarcinus maenas to hypercapniaTable S2. Details on primers for quantitative real-time polymerase chain reaction (qRT-PCR) to assess responses of . Primer sequences (5' → 3') and descriptions of the targeted genes used in the real-time polymerase chain reaction (qRT-PCR) in the short- and long-term hypercapnia experiments on Carcinus maenas. Numbers ('no.') are according to the numbers used in Figure 4 and Additional File C. maenas microarray by Towle et al. [2 and efficiency were tested in a qRT-PCR dilution series . e et al.  and the Carcinus maenasTable S3. Transcripts significantly regulated in response to hypercapnia in . Transcripts significantly regulated in response to hypercapnia in Carcinus maenas as identified by variance-based linear modelling (F-test) and sign test. Out of 1634 genes, 678 were identified by sign test, 578 genes were identified by F-test, and 378 were identified to be affected by both statistical tests. Transcripts are sorted alphabetically after the accession number ). Bold and underlined = significantly regulated as identified by sign test, p < 0.05; bold = significantly regulated as identified by both statistical tests ; italic = differentially expressed as identified by F-Test. Accession numbers refer to the ESTs generated for the C. maenas microarray by Towle et al. [Carcinus maenas when exposed to hypercapniaTable S4. Significantly regulated transcripts associated with a cellular stress-response in . Significantly regulated transcripts in the short-term hypercapnia study on Carcinus maenas associated with a cellular stress-response acoording to [rding to . Bold anCarcinus maenasTable S5. Significantly up-regulated transcripts in the short-term hypercapnia study associated with structural molecule activity in gills of . Transcripts identified to be responsible for the over-representation of the GO-term "structural modification" (GO:0005198) in Carcinus maenas gills exposed to hypercapnia . Bold and underlined  = significantly regulated as identified by sign test (p < 0.05); bold (ACC. no.) = identified to be significantly regulatd by both tests . Accession numbers (ACCs) refer to the database GenBank (NCBI). Carcinus maenas after exposure to short-term hypercapniaTable S6. Details on transcripts of special interest identified by microarray analysis on gills of . Identification and details on transcripts of special interest identified by microarray analysis, after an additional NCBI blastx. * = NCBI blastn; ** = see Towle et al. [e et al.  for detaClick here for fileCarcinus maenas to short-term hypercapniafigure S1. Comparison of distinct transcripts of the microarray analysis vs. qRT-PCR for the response of . Comparison of the regulation of distinct transcripts of gill 9 for the Carcinus maenas response to short-term hypercapnia  in the microarray analysis with results of the qRT-PCR experiment performed on the respective genes from the short-term incubation conducted in April 2010. In 7 of 8 cases, both techniques show the same tendency in regulation. Values represent median log2-ratios with median deviation (error bars). Transcript numbers according to Additional File +/K+-ATPase alpha subunit, Cl-channel (Ca-act.) = calcium acitvated chloride channel.Click here for file


The precise mechanisms of the neuroprotective effects of insulin in streptozotocin (STZ)-induced diabetic animals remain unknown, but altered peripheral nerve insulin receptor signaling due to insulin deficiency might be one cause.p = 0.0351) in the I-group compared with the U-group. Western blots showed unaltered total insulin receptor (IR), but a 31% decrease and 3.1- and 4.0-fold increases in phosphorylated IR, p44, and p42 MAPK protein levels, respectively, in sciatic nerves from the U-group compared with the C-group. Phosphorylated p44/42 MAPK protein decreased to control levels in the I-group (p<0.0001).Diabetes was induced in 10-week-old, male Wistar rats by injecting them with STZ (45 mg/kg). They were assigned to one group that received half of an insulin implant  or another that remained untreated  for 6 weeks. The controls were age- and sex-matched, non-diabetic Wistar rats . Low-dose insulin did not change haemoglobin A1c, which increased by 136% in the U-group compared with the C-group. Thermal hypoalgesia and mechanical hyperalgesia developed in the U-group, but not in the I-group. Sensory and motor nerve conduction velocities decreased in the U-group, whereas sensory nerve conduction velocity increased by 7% (Low-dose insulin deactivated p44/42 MAPK and ameliorated peripheral sensory nerve dysfunction in rats with STZ-induced diabetes. These findings support the notion that insulin deficiency per se introduces impaired insulin receptor signaling in type 1 diabetic neuropathy. Insulin acts as a potent neurotrophic factor that specifically stimulates neurite outgrowth and regeneration of sensory neurons , they were assigned to one group that received half of an insulin implant (∼1 U/day)  (n = 11) or another that remained untreated (n = 10) for 6 weeks. The implant was placed in the subcutaneous tissue located on the back following the manufacturer's instructions (http://www.linshincanada.com/linbit.html) under anaesthesia with pentobarbital (40 mg/kg). The controls were age- and sex-matched, non-diabetic, Wistar rats (n  = 12). All animals were housed in sawdust in plastic cages. The rats were maintained on a 12-h/12-h, light/dark cycle at 22°C±2°C and 55%±5% relative humidity and allowed free access to tap water and standard laboratory chow . The animals were weighed, and blood glucose levels were monitored regularly. Whole blood glucose levels of tail vein samples obtained between 14:00 and 16:00 were measured using an Accu-Chek Compact Plus blood glucose meter . At the end of the experiment, non-fasting animals were killed by exsanguination from the left cardiac ventricle under deep anaesthesia with pentobarbital (∼100 mg/kg) between 10:00 and 16:00. Then, blood samples were collected to determine hemoglobin A1c, serum lipid, insulin, adiponectin, and leptin levels, as described previously All animal protocols were approved by the Animal Research Committee and followed the Guidelines for Animal Experimentation of Hirosaki University (M08088) as well as the Principles of laboratory animal care . All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Diabetes was induced in 10-week-old, male Wistar rats  by injecting them with STZ (45 mg/kg). Six weeks after the induction of diabetesBefore starting the experiments, the animals were allowed to acclimatize to handling-related stress for at least 1 week. At the indicated time points, tail-flick latency (TFL) and the mechanical nociceptive threshold (MNT) of each animal were determined to assess nociceptive responses to noxious thermal and pressure stimuli, respectively. The MNT was assessed with the Randall-Selitto test using an Analgesymeter . A constantly increasing pressure stimulus (with increase at a rate of 16 g/s) was applied to the dorsal surface of the rat hind paw while the animal was gently restrained under a soft towel; to avoid tissue damage, a cutoff of 250 g was used. The pressure was increased until the animal withdrew the paw, squeaked, or struggled. One measurement per paw was performed with an interval of longer than 15 minutes between measurements; for each animal, the results for the 2 paws were averaged for use in statistical analysis. Thermal sensitivity was assessed using a Tail Flick Analgesymeter . The animal was gently wrapped in a towel and placed on the top of the instrument with the tail in the sensing groove. The tail flick latency was determined by exposing the animal’s tail to a radiant heat source and recording the time taken to remove the tail from the noxious thermal stimulus. The radiation intensity was chosen on the basis of the intensity required to elicit a basal tail flick response of 4 to 8 seconds in control rats. For each animal, 2 to 3 recordings were made at an interval of longer than 15 minutes; the mean value was used for statistical analysis.At the end of the experiment, nerve conduction was assessed in sciatic-tibial nerves as described Sural nerves at the level of the lower calf were excised and subjected to morphometric analyses as described previously Hind footpad skin specimens were obtained with a 3-mm punch tool and processed as described previously Bilateral lumbar DRGs (L4) were excised at the end of the experiment and processed as described previously The left sciatic nerve was collected from each animal and subjected to Western blot analysis as described previously p value of <0.05 was considered significant.All data were statistically analysed using Stat View software (version 4.5). Values are described as means ± SE. The significance of differences in mean values between two groups was tested by ANOVA followed by the Bonferroni/Dunn test. A two-sided At the end of the experiment, the body weight of untreated STZ-induced diabetic rats was less than that of control rats and comparable to that of insulin-treated STZ-induced diabetic rats . Hemoglop<0.05) and 18% (p<0.05), respectively, whereas MNT to pressure stimuli (p > 0.05) and 16% , respectively, compared with control rats. Low-dose insulin administration for 4 to 5 weeks reversed the thermal and mechanical pain sensations.Six weeks after diabetes was induced in untreated and insulin-treated STZ-induced diabetic rats, TFL to thermal stimuli  was incr stimuli  was decrp<0.05) by 7% and MNCV increased non-significantly (p = 0.07) by 6% compared with untreated STZ-induced diabetic rats.In untreated STZ-induced diabetic rats, both SNCV  and MNCVFascicular area, myelinated fiber density, myelinated fiber area, axonal area, and the axon/myelin ratio of the sural nerves were not significantly different among the control, untreated and insulin-treated STZ-induced diabetic rats . The sizThe IENFD tended to increase by 21% in the footpad of untreated STZ-induced diabetic rats compared with control rats; it was not affected by low-dose insulin administration .2 in control rats , the p38 kinases, and the c-jun N-terminal kinases (JNKs), and their pathways may be activated in injured nerves via distinct molecular and cellular mechanisms 2 in untreated STZ-induced diabetic rats, which was partially restored by insulin treatment  pathway activity, reduced myo-inositol content, altered protein kinase C activity, and oxidative stress, have been claimed to be central to the pathogenesis of diabetic neuropathy. Based on this assumption, a number of clinical intervention trials have been performed. However, our understanding of human diabetic neuropathy remains incomplete. In this regard, peripheral nerve dysfunction in STZ-induced diabetic animals may correlate with insulin deficiency Quantification of intraepidermal nerve fibers has increasingly been used to evaluate the extent of involvement of unmyelinated sensory fibers In summary, insulin-deficient STZ-induced diabetic rats had distinct alterations in peripheral nerve insulin receptor signaling that differed from those in insulin-resistant ZDF rats. A low dose of insulin, insufficient to affect systemic glycemia, partially restored the impaired peripheral nerve insulin receptor signaling involving the deactivation of p44/42 MAPK and ameliorated peripheral sensory nerve dysfunction in STZ-induced diabetic rats. These findings support the notion that, besides hyperglycemia, insulin deficiency is involved in the pathogenesis of type 1 diabetic neuropathy.


Background/Objective. It is known that menopause or lack of endogenous estrogen is a risk factor for endothelial dysfunction and CAD. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved inmultiple phases of vascular dysfunction.The purpose of the current study was to determine the association between soluble LOX-1 (sLOX-1) and pregnancy followed by delivery in women of reproductive age. Materials/Methods. Sixty-eight subjects with pregnancy followed by delivery (group 1) and 57 subjects with nongravidity (group 2) were included in this study. Levels of sLOX-1 were measured in serum by EL SA. Results. Plasma levels of sLOX-1 were significantly lower in Group 1 than Group 2 in women of reproductive age . There were strong correlations between sLOX-1 levels and the number of gravida . The levels of sLOX-1 highly correlated with the number of parous .  Conclusion. Our study demonstrated that serum sLOX-1 levels were associated with pregnancy followed by delivery that might predict endothelial dysfunction. We conclude that pregnancy followed by delivery may delay the beginning and progress of arteriosclerosis and its clinical manifestations in women of reproductive age. Even though substantial efforts have been made to improve education and public awareness and despite the use of effective medications and life-style changes for controlling the associated risk factors, coronary artery disease (CAD) remains the leading cause of death in women worldwide , 2. In cDuring normal menstrual cycles, women show high levels of estrogen just before ovulation and during the luteal phase and in the normal physiology of pregnancy, women have significantly higher levels of estrogen derived mainly from the placenta . EstrogeLectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a type II membrane glycoprotein, is the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells . OxidatiTherefore, we hypothesized that if women have been exposed for a longer time and/or at a higher level to endogenous (not exogenous) estrogen, such as pregnancy followed by delivery and/or gravidity, they may obtain estrogen's beneficial “cardioprotective,” “antiatherosclerosis,” and/or “antioxidant” effect. The purpose of the current study was to determine the association between pregnancy followed by delivery and sLOX-1.From January 2010 to June 2011, we prospectively evaluated 1284 patients in cardiology outpatient clinic of our hospital. Sixty-eight subjects with pregnancy followed by delivery (Group 1) and 57 subjects with nongravidity (Group 2) were included in this study. All participants provided written informed consent to participate in the study. The protocol was approved by the local Ethics Committee.Exclusion criteria included pregnancy, known polycystic ovary syndrome, congestive heart failure (ejection fraction <50%), myocardial infarction, stroke, known peripheral atherosclerotic disease, surgical coronary intervention, other major vascular surgical procedures, coronary angioplasty, unstable angina pectoris, diabetes mellitus, hypertension, suspected myocarditis or pericarditis, impaired renal function (creatinine ≥1.4 mg/dL), unstable endocrine or metabolic diseases known to influence serum inflammation markers, concomitant inflammatory diseases such as infections and autoimmune disorders, active or chronic hepatic/hepatobiliary disease, and malignancy. Patients taking oral contraceptive, corticosteroids, anti-oxidant vitamins, and alcohol were also excluded from the study.Blood samples of all individuals were taken from an antecubital vein following an overnight fasting state at the first three days of menarche. After centrifugation at 3000 ×g for 10 minutes, serum and plasma samples were frozen and stored at −80°C until an assay could be performed. Serum sLOX-1 levels were measured by a commercially available enzyme-linked immunosorbent assay kit . The detection limit for serum sLOX-1 level was 2.4 pg/mL with a coefficient of variation <5%. Triglyceride (TG), total cholesterol (tot-C), LDL-C, and high-density lipoprotein cholesterol (HDL-C) concentrations were measured by automated chemistry analyzer  by using commercially available kits.t-test. Correlation analyses were performed using the Pearson coefficient of correlation. SPSS 15.0 software was used for basic statistical analysis . A value of P < 0.05 was accepted as statistically significant.Continuous variables were given as mean ± SD; categorical variables were defined as percentages. Comparisons between Group-1 and Group-2 were carried out using an independent samples P = 0.1). The mean age of first menarche was 12.1 ± 2.3 years in pregnancy followed by delivery group and 11.9 ± 1.9 years in nongravidity group (P = 0.5). The rates of family history and smoke were similar between the two groups (P = 0.1). The levels of total-C, LDL-C, HDL-C, and triglyceride were also similar between the two groups . The sLOX-1 levels highly negatively correlated with the number of gravida . r = −0.683, P < 0.001). The sLOX-1 levels were not correlated with age and age of first menarche . In the multiple linear regression analysis age was positively related and number of gravity was negatively related with sLOX-1 levels .To the best of our knowledge, this is the first study that shows the relationship between sLOX-1 levels and pregnancy followed by delivery in women of reproductive age. This study showed that women who had at least 1 pregnancy followed by delivery showed a decreased level of sLOX-1 compared with those who had never experienced delivery. The sLOX-1 levels had significantly negative correlation with gravida and parous. We hypothesized that if women have been exposed for a longer time and/or at a higher level to endogenous (not exogenous) estrogen, such as pregnancy followed by delivery and/or gravidity, they may obtain estrogen's beneficial effect and may have a greater decrease in level of sLOX-1. These findings may support the idea that as long as women are exposed to endogenous estrogen they have decreased level of sLOX-1.Coronary artery disease remains the leading cause of death in the 21st century. Despite the advances in this area, it is still the main cause of death among women in developed countries . The preThe well-known risks for CAD, such as systemic hypertension, smoking, obesity, sedentary life-style, dyslipidemia, stress, family history of CAD, diabetes mellitus, menopause, lack of endogenous estrogen, and insulin resistance, are numerous . More re The incidence of CAD and mortality is very low in women of reproductive age but rises to a significant level in menopause women . There iβ-estradiol-based preparations have clearly shown decreased LDL-C oxidation, and in addition, estradiol reduces the development of early lesions of atherosclerosis, in part through the effects on lipid metabolism which reduce lipid deposits in the endothelium [Disturbances in endothelial function have an important role in the physiopathology of atherosclerosis, and several lines of evidence suggest that interventions in endothelial function could modify the progress rates of atherosclerotic disease and the risk of cardiovascular events. Some studies have documented that estrogens are potent antioxidants and decrease LDL-C oxidation in vitro and in vivo , 13. Stuothelium , 37.β-estradiol were used as an antiatherosclerogenic agent to demonstrate feasibility in an in vitro vascular model [Cevic et al. demonstrated that a high concentration of estrogen reduces the level of asymmetric dimethylarginine (ADMA), which is an endogenous competitive inhibitor of NO synthase . Estradiar model .LOX-1, a type II membrane glycoprotein, is the major receptor for ox-LDL in endothelial cells . It is a It is well known that menopause or lack of endogenous estrogen is a risk factor for cardiovascular disease –51. HashIn conclusion, our study demonstrated that serum sLOX-1 levels were associated with pregnancy followed by delivery which might predict endothelial dysfunction. Pregnancy followed by delivery may improve endothelial function and prevent the progress of atherosclerosis in women of reproductive age. LOX-1 can be used as a target for imaging of endothelial function. We conclude that pregnancy followed by delivery may delay the progress of arteriosclerosis and its clinical manifestations in women of reproductive age.


MircoRNAs (miRNAs) are small non-coding RNAs that govern the gene expression and, play significant role in the pathogenesis of heart failure. The detection of miRNAs in circulation of pulmonary hypertensive (PH) human subjects remains elusive. In the current study, we determined the pattern of miRNAs of mild-to-severe human PH subjects and, compared them with the control subjects by miRNA array. Blood was obtained using fluoroscopic and waveform guided catheterization from the distal (pulmonary artery) port of the catheter. A total 40 human subjects were included in the study and, the degree of PH was determined by mean pulmonary arterial pressure. Among several miRNAs in the array, we validated 14 miRNAs and, the data were consistent with the array profile. We identified several novel downregulated miRNAs  and upregulated miRNAs  in the circulation of PH subjects. Our study showed novel set of miRNAs which are dysregulated in PH and, are directly proportional to the degree of PH. These miRNAs may be considered as potential biomarker for early detection of PH. MicroRNAs (miRNAs) are small, endogenously expressed noncoding RNAs that regulate gene expression at post-transcriptional level, via degradation or translational inhibition of their target mRNAs Pulmonary hypertension (PH) is a progressive pulmonary vascular disease with high morbidity and mortality. PH results in right ventricular hypertrophy (RVH), progressive fibrosis and RV failure; and low cardiac output leading to increase morbidity, and mortality Recent studies have shown that miRNAs are circulating freely in the mammalian blood and can be predicted as “biomarker” for early diagnosis of acute myocardial infarction and heart failure in humans The patients were visited at Scott & White Hospital, Temple, TX with PH diagnosed by right heart cath. The consent was obtained from all participants before enrolled in this study. Forty consecutive patients undergoing right heart catheterization for diagnosis of pulmonary hypertension or monitoring of disease progression and response to therapy were enrolled in the study. The study protocol was approved by the ethics committee of Scott & White Hospital that conforms to the principles outlined in the Declaration of Helsinki. All clinical samples were obtained with approval of the Scott & White Hospital research ethics committees. The written consents were obtained from each patient for this study and were kept on file. The consent procedure was approved by S & W Ethics' committee. The protocol (#100029) was approved by the Scott & White Healthcare Institutional Review Board.After fluoroscopic and waveform guided catheterization of the pulmonary artery, approximately 20 ml of blood was obtained from the distal (pulmonary artery) port of the catheter with the balloon deflated. The blood was then injected into purple top tubes and transported to the laboratory for processing as described below.Patient characteristics are summarized in Of the patients included in group 1, 4 were treatment naïve, 3 were taking calcium channel blockers alone, 2 were taking sildenifil and ambrisentan, 1 was taking bosentan and calcium channel blockers, and 1 was taking tadalafil and calcium channel blockers.The miRNA array was performed using the PH subject whose mean PAP was 53 mm Hg and diagnosed as idiopathic pulmonary hypertensive patient. The samples we validated in our cohort study were considered as pulmonary hypertensive. However, these subjects were also exhibited symptoms associated with PH like COPD, interstitial lung disease, obstructive lung disease, scleroderma, etc. There was no morbidity occurs so far in this study group; therefore, we believe that the changes of miRNA in the blood were associated with PH.During the right heart catheterization, approximately twenty milliliters of blood were obtained from the pulmonary artery, processed and, the buffy coats were prepared and were stored at −80°C.The miRNA was isolated using the PH subject whose mean PAP was 53 mmHg and diagnosed as idiopathic pulmonary hypertensive patient. The control subjects miRNA was isolated from the subjects whose mean PAP was 20 mmHg. Isolation of miRNA was done using miRNeasy Mini Kit  as per manufacturer's instructions. Briefly, Twenty milliliters of blood were collected from study subjects in PAXgene Blood RNA tubes . The buffy coats (250 µL each) were homogenized in 700 µL of QIAzol lysis reagent. After addition of 140 µL chloroform, the homogenate was separated into aqueous and organic phases by centrifugation. The upper aqueous phase (approximately 320 µL) containing the RNA was extracted, and 100% ethanol (approximately 525 µL) was added to provide appropriate binding conditions for all RNA molecules from 18 nucleotides (nt) upwards into the miRNeasy columns. This was then applied to the RNeasy Mini spin column, where the total RNA was bound to the membrane and phenol and other contaminants were washed away by washing with the wash buffer (700 µl Buffer RWT) and subsequently, twice with 500 µl of Buffer RPE. Total microRNA was then eluted in 20 µL RNase-free water.2 value. Paired t-test or ANOVA were performed to find statistically significant difference between the groups and p<0.05 was considered as a significant.RNA was quantified and assessed using the RNA 6000 Nano Kit and 2100 Bio analyzer . MiRNA array was performed and analyzed by LC Bioscience, Austin, TX, USA. In brief, this analysis was performed on the Agilent miRNA array, using the labeling kit, reagents and methods recommended by the manufacturer. Samples for miRNA profiling studies were processed by LC Bioscience , according to standard operating procedures in the GLP-compliant services laboratory. Total RNA is first dephosphorylated and then the pCp-Cy3 labeling molecule is ligated to the 3′ ends of the RNA molecules. The labeled RNA is purified using BioSpin6 . Hybridization, washing, staining, imaging, and signal extraction were performed according to Agilent-recommended procedures. The data represent as logAverage values of the replicate spots of each miRNA on the microarray were normalized using global normalization. The correction factor was calculated by dividing the sum of intensities of each sample by the average of all the samples. The normalized values were calculated by multiplying average intensities of each miRNA with the correction factor. The JMP 6.0.0 software  was used for hierarchical clustering, generation of heat maps, and principal component analysis (PCA). The miRNA array data is MIAME compliant has been submitted to the Gene expression Omnibus server at NCBI vide GEO no GSE4415.The samples we validated in our cohort study were considered as pulmonary hypertensive. However, these subjects were also exhibited symptoms associated with PH like COPD, interstitial lung disease, obstructive lung disease, scleroderma, etc. There was no morbidity occur so far in this study group, therefore, we believe that the changes of miRNA in the blood were associated with PH.2 miRNA First Strand Kit . The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added to individual specific primers and, has- miR-U6 (Housekeeping micro-RNA) as per manufacturer's instruction in technical duplicates. PCR was performed on a Stratagene MX 3005 Real-Time PCR System . The following thermal cycle was used 95°C, 10 min, 40 cycles of . ROX was used as internal reference dye.To confirm the miRNA array data, we performed a qRT-PCR using buffy coat. A total of 40 samples were studied as a cohort basis. Two hundred nano-grams of each subjects RNA enriched for small species with length <200 bases were reverse-transcribed into first strand cDNA using an RTCt was normalized using the quintile method to remove the variations from the housekeeping genes. The fold change for each miRNA was calculated using ΔΔCt method. The data for the expression of these miRNAs was normalized to 106 copies of housekeeping has-miR-U6. If the expression of miRNAs of interest showed a copy number of ≤1 per 104 copies of has-miR-U6, it was termed as “not detected.”Threshold cycle (Ct) for each miRNA was extracted using MXPro software (Stratagene) by setting threshold and automatically defined baseline. The raw Statistical analyses were performed using GraphPad Prism 5.0 statistical package for Windows . Data are presented as mean ± SEM unless indicated otherwise and, plasma miRNA levels are presented as fold-change relative to the healthy subjects. Linear regression analyses were used to correlate quantitative variables after normalization of non-Gaussian variables by log transformation. Comparisons between 2 groups were performed using Student t tests for Gaussian data or Mann-Whitney tests for non-Gaussian data. The P-value (P<0.05) was considered as statistically significant differences between the two groups. Results were also analyzed by one-way ANOVA for comparative analysis in between the groups (moderate and severe PH human subjects) with those of healthy control subjects.We have included mean pulmonary artery pressure (mPAP), right atrial pressure (RA), pulmonary vascular resistance (PVR), pulmonary artery systolic pressure (PAS) and pulmonary artery diastolic pressure (PAD) for moderate-to-severe PH in human subjects. Our data showed that mPAP, PAS and PAD were significantly increased in the PH patients compared to the controls . Additio2- transformed, balanced) and the p values of the t-test were calculated.This is the first report where we observed differential miRNA expression in patients with PH and compared with healthy individuals who did not show the mean arterial pressure greater than 25 mm of Hg by miRNA microarray. We observed that several miRNAs were differentially expressed in the PH patients compared to control subjects . The datWe validated a set of miRNAs in our cohort study. We choose the following miRNAs for validation:2 and, miR-130a has target for BMPR1b and SMAD2. It is of note that these miRNAs were not detected in the miRNA array possibly because of low level of detection limit.MiR-21, miR-23a, miR-26a, miR-29, miR-34b, miR-191, miR-451 and miR-1246 were derived from the miRNA array analysis . We alsovs. severe PH.We determined the expression of circulatory miRNAs in control, moderate PH and severe PH subjects. The expressions of miR-26a, miR-29c, miR-451 and miR-1246 were declined to 0.66±0.12, 0.52±0.14, 0.49±0.16 (p<0.05) and 0.67±0.21-folds (p = ns) respectively, in moderate PH subjects, compared to the control subjects. The expressions of the above miRNA were further declined to 0.49±0.18, 0.42±0.14, 0.34±0.14 and 0.36±0.17-fold respectively (p<0.05), in severe PH subjects, compared to the control subjects . The expThe expression of miR-130a, miR-133b and miR-191 were increased significantly in all PH subjects and, showed further pronounced in severe PH subjects. The expression of miR-130a showed a 2.54±0.56-fold (p<0.05) enhancement in moderate PH where severe PH showed a sharp increment of 9.27±1.19-fold (p<0.01), respectively. In moderate PH subjects, the expression of miR-133b was 2.70±1.07-fold and 3.96±1.63-fold (p = ns) in moderate and severe PH subjects, compared to the control subjects. Likewise, in the moderate PH group, the expression of miR-191 was 2.88±0.71-fold and was further increased to 5.83±1.66-fold (p<0.05) in severe PH subjects, compared to the controls . The expThe expression of miR-21 and miR-23b were moderately increased in all PH subjects but showed more pronounced in severe PH subjects. In moderate PH subjects, the expression of miR-21 was 2.4±0.36-fold and 3.09±0.71-fold (p<0.05) in severe PH subjects, compared to the control subjects. Likewise, in the moderate PH group, the expression of miR-23b was 2.39±0.76-fold (p<0.05) and 4.07±0.88-fold (p<0.01) increased in severe PH subjects, compared to the controls . The expIn order to determine whether gender has any influence in circulatory miRNA expression, we determined all validated miRNAs as gender based cohort study. We compared the miRNA expressions pattern in male and female PH subjects separately; and both the genders . Among tetc, and, may therefore, consider as an idiopathic type. However, our data are consistent with respect to mPAP elevation and severity of PH which is the hallmark for PH. The observation may provide early detection of PH in human serum.The present study for the first time demonstrates that PH induces novel circulatory miRNA derived from human PH subject. Recent evidences indicate that freely circulating miRNAs may be informative in the diagnostic setting and have the potential to be used as biomarkers. Our study demonstrates that in human subjects with diverse clinical settings in PH are associated with distinct dysregulation of miRNAs in the circulation. It is of note that PH is associated with other diseases like COPD, scleroderma, obstructive lung diseases, Our study reveals that in human subjects with moderate to severe PH are associated with significant downregulation of plasma levels of circulatory miR-1, miR-26a and miR-29c. The kinetics of the changes differed from moderate to severe PH human subjects as we observed further downregulation of the above miRNAs in severe PH. Targetscan analysis showed that miR-26a and miR-29c had target genes for collagens which involved in pro-fibrotic events and fibrosis. It is of note that the patient population include in this study suffered from lung fibrosis, interstitial lung disease and, RVH that lead to cardiac fibrosis. The miR-1 has target genes for Notch3 which is upregulated in PH in human as well as in rat model may indicate a functional correlation et al showed the downregulation of miR-204 in human PH subjects and in experimentally induced PH rat model and discovered a new regulatory pathway involving miR-204, which was critical to the etiology of PH. The study showed that delivery of synthetic miR-204 to the lungs of animals with PH significantly mitigated disease severity Our data indicated a set of upregulated miRNA in the circulation of PH subjected compared, to the controls. During moderate PH, miR-21, miR-23b, miR-130a, miR-491 and miR-1246 are moderately upregulated but, are more pronounced in severe PH, compared to the controls. Our data also revealed another set of miRNAs that include miR-133b, miR-204 and miR-208b which were significantly upregulated in all moderate PH subjects but, showed further increment in severe PH subjects. Among them, miR-208b and miR-133b are considered to be cardiac specific suggesting that they may be released from the injured non-cardiac muscles Together, these data indicated a potential use of these miRNAs as an early detection biomarker of PH. From this study, we present evidence that a set of increased plasma miRNA level that include miR-21, miR-130a, miR-133b, miR-191, miR-204 and miR-208b, can be used as biomarker for assessment of PH. On the contrary, our data revealed a set of declined miRNAs which includes miR-26a, miR-29c, miR-34b and miR-451and; can be used as biomarker as well. These findings are of vital importance for the diagnostic applicability of plasma miRNAs in clinical settings and in particular, where altered distributions of biomarkers, combined with co-morbidities such RVH, pulmonary fibrosis, are common. Alternatively, the significant decrease of miR-1, miR-26a and miR-29c level in PH has potential diagnostic significance.In summary, the current study provides for the first time the detection of circulatory miRNAs in PH human subjects. We report that initial PH induced damage of endothelial cells marked by increased mPAP and RA initiates dysregulation of miRNAs in the organ or tissue architecture which then shedding off and, come into the circulation. Our study suggests that a specific set of dysregulation (up- and down regulation) of miRNAs originating from either cardiac or vascular bed can be detected in the circulation. Our study includes a diverse PH cohort, but, it is noted that all subjects are diagnosed with PH. We, therefore, assume that changes of miRNA in circulation are primarily due to elevated PH. The dysregulation of these miRNAs may serve as therapeutic target for drug invention or early diagnostic tool for PH, PH induced RVH and interstitial lung disease. Further experimental studies are required to define the synergistic association between the tissue expressions versus circulation in patient population. Although circulatory miRNA offers a great potential for the diagnosis; however, precise quantification, normalization and larger cohort studies are warranted. These parameters are necessary for accurately translating the findings into clinical biomarkers.Our study provides the first evidence of circulating miRNAs in the setting PH may use as an early detection for PH. Nevertheless, the data presented here are based on limited study population and, the results need to be confirmed in a larger cohort study. Furthermore, the dysregulation miRNA in PH remains to be tested whether these circulatory miRNAs have any influence in the target genes. Elucidating the possible biological role of PH specific miRNAs in the circulation is beyond the scope of this study. However, considering the recent reports describing cell-to-cell transport of miRNAs Supporting Information S1Patients Profile.(PDF)Click here for additional data file.


Adipocyte fatty acid-binding protein (A-FABP) has been described as a novel adipokine, playing an important role in the development of metabolic syndrome, type 2 diabetes and atherosclerosis. In this study, we investigated the relationship between serum levels of A-FABP and the presence and severity of coronary artery disease (CAD) in Chinese subjects.P = 0.029 in men; P = 0.031 in women). Serum A-FABP increased significantly in multi-vessel diseased patients than in non-CAD subjects , and showed an independent correlation with coronary atherosclerosis index . In multiple logistic regression analysis, serum A-FABP was an independent risk factor for CAD in women . In addition, amino terminal pro-brain natriuretic peptide (NT-proBNP) was demonstrated to be positively and independently correlated with A-FABP .Circulating A-FABP level was determined by ELISA in 341 Chinese subjects  who underwent coronary angiography. A-FABP levels in patients with CAD were significantly higher compared with non-CAD subjects (Serum A-FABP is closely associated with the presence and severity of CAD in Chinese women. Adipose tissue functions as an endocrine organ, secreting a variety of bioactive substances , which are actively involved in insulin sensitivity, glucose and lipid metabolism -/-) mice, A-FABP deficiency reduced early foam cell formation greatly, and provided remarkable protection against atherosclerosis with significant reductions in mean atherosclerotic lesion area on a chow diet or a western diet independent of the effects of A-FABP on insulin resistance and plasma lipids -/- mice There is substantial experimental evidence that A-FABP plays an important role in metabolic deterioration and the development of atherosclerosis P = 0.096 and P = 0.088) Although originally identified as a major cytoplasmic protein, A-FABP is also released from adipose tissue into the circulation All subjects gave written informed consent, and the study was approved by the ethics committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital and complied with the Declaration of Helsinki.The study population consisted of 341 participants  who were admitted to the Department of Cardiology of Shanghai Jiao Tong University Affiliated Sixth People's Hospital to undergo coronary angiography between July 2008 and October 2009. All women were postmenopausal. The diagnosis of type 2 diabetes was based on 1999 World Health Organization criteria Subjects with the following conditions were excluded: hepatic or renal dysfunction, recent myocardial infarction (<3 months), previous coronary by-pass surgery or percutaneous coronary intervention (<6 months), congestive heart failure (New York Heart Association Class III∼IV), acute infection, and history of malignancy.Coronary angiography was performed using standard Judkins techniques The extent and severity of CAD was assessed by assigning points to each lesion as follows: no significant stenosis of the luminal diameter, 0; stenosis of less than 25%, 1; 25–49% stenosis, 2; 50–74%, 3; 75–100%, 4. The coronary atherosclerosis index (CAI) was defined as the sum of these scores 2). Waist circumference was measured midway between the lowest rib and the superior border of iliac crest on midaxillary line.A complete physical examination was performed on each subject. Body mass index (BMI) was calculated as weight in kilograms divided by squared height in meters . Glycated hemoglobin A1c (HbA1c) values were measured by high performance liquid chromatography . Serum creatinine and lipid profiles, including total cholesterol (TC), TG, HDL-c and low density lipoprotein cholesterol (LDL-c) were determined by enzymatic procedures using an autoanalyser . Serum hsCRP was measured by a particle-enhanced immunoturbidimetric assay . NT-proBNP was assayed by chemiluminescence . A-FABP levels were assayed by sandwich ELISA . Insulin resistance was estimated using homeostasis model assessment index (HOMA-IR) P<0.05 were considered as statistically significant.All analyses were performed using SPSS version 13.0 . Data were reported as means ± SD for normal distributions or median with interquartile range for skewed variables. Data that were not normally distributed as determined by the Kolmogorov-Smirnov test were logarithmically transformed before analysis. Characteristics of subjects between non-CAD and CAD groups or men and women were compared using unpaired Student's t-test  or the Mann-Whitney U-test . One-way ANOVA was used to compare serum A-FABP levels in groups according to number of diseased vessels. The chi-square test was used to compare categorical variables between groups. The Spearman correlation analysis was used to assess the correlations of CAI and serum A-FABP with metabolic parameters. To determine the independent parameters correlated with CAI and A-FABP, parameters including age, gender, and those correlated significantly with CAI and A-FABP in correlation analysis were tested in multiple stepwise regression analysis. Multiple logistic regression analysis was performed to identify independent predictors for CAD. Parameters that significantly different between subjects with and without CAD and conventional risk factors including lipid profile  were selected to enter into multiple logistic regression. Two-sided values of P<0.05). Women with CAD also had significant higher systolic blood pressure, 2hPG and the percentage of hypertension than non-CAD controls .The clinical characteristics of the subjects are shown in P<0.001) and non-CAD group (14.4 ng/ml (7.4–22.9) vs. 6.5 ng/ml (4.4–10.7), P<0.001). Also, A-FABP levels in patients with CAD increased significantly compared with non-CAD subjects in both men and women .As shown in P<0.001). After adjusting for age and gender, CAI showed a positive correlation with HbA1c, fasting insulin, HOMA-IR, NT-proBNP and serum A-FABP (P<0.05), and an inverse correlation with HDL-c (P = 0.029) (P = 0.025). Furthermore, as shown in P = 0.008 in men and P = 0.004 in women). Compared with those without CAD, patients with multi-vessel disease had significantly higher serum A-FABP levels in both men (P = 0.011) and women (P = 0.004). In addition, significant higher levels of A-FABP were also observed in women with multi-vessel disease compared with those with 1 vessel disease (P = 0.031).We next analyzed the relationship between serum A-FABP and the severity of CAD. In all subjects, CAI was positively associated with age  . In multP = 0.021) and systolic blood pressure  were demonstrated to be independent factors for CAD in women. However in men, only age was independently associated with the presence of CAD .To evaluate the relationship between A-FABP and CAD, multiple logistic regression analysis was performed using the presence of CAD as a dependent variable . The anaP<0.05) (P<0.05). To further determine which variables were independently correlated with serum A-FABP, multiple stepwise regression analysis was performed in all subjects (P<0.05).A-FABP showed significant positive correlations with age, BMI, waist circumference, 2hPG, HOMA-IR, creatinine, hsCRP, and NT-proBNP, and a negative correlation with GFR in both genders  . A-FABP subjects . The anaIn the current study, we provided the clinical evidence showing that A-FABP was closely associated with the severity of coronary atherosclerosis, and was a significant risk factor for the development of CAD in women. Furthermore, we found a close relationship of serum A-FABP with NT-proBNP in both genders.The relationship of A-FABP with metabolic disease has been well demonstrated in several clinical studies, which might partly explain the impact of A-FABP on CAD. Xu et al. demonstrated that A-FABP levels were not only associated with insulin resistance, adiposity, dyslipidemia and glucose tolerance parameters, but also predicted the development of the metabolic syndrome and type 2 diabetes in 5-year and 10-year prospective studies -/- mouse model on a western diet In addition to its metabolic actions, A-FABP also plays a critical role in the development of atherosclerosis through coordinating macrophage cholesterol trafficking and inflammatory activity 2+ levels Another notable observation of this study is the independent association between serum A-FABP and NT-proBNP, a hormone secreted by cardiomyocytes following increased myocardial wall stress and exerts favorable effects on heart such as promoting vasodialation In the present study, we also found an independent negative relationship between A-FABP and renal function as assessed by GFR and serum creatinine, which is in accordance with a recent study This study has several limitations. First, the sample size was not large enough. Second, the present study is cross-sectional, which does not allow us to determine the cause-effect relationship between A-FABP and the development of CAD. Further large population-based prospective studies are needed to address whether serum A-FABP is useful for predicting CAD. Furthermore, in order to obtain more accurate data about coronary stenosis, we only recruited subjects admitted to hospital to undergo coronary angiography, and most of these people have existing risk factors of CAD, or already on intervention therapy. Whether our findings can be replicated in the general population still needs to be further confirmed.In conclusion, we demonstrated that serum A-FABP was closely associated with the presence and severity of CAD in Chinese women. A-FABP may be an independent marker and predictor of CAD. Our data, in conjunction with previous animal and clinical studies, support the central role of A-FABP in the development of the metabolic syndrome, diabetes and cardiovascular disease. Further studies are warranted to evaluate whether A-FABP can be used to predict the risk of CAD in larger study cohorts recruited from the general population.


To elucidate the molecular basis of early gastric cancer (EGC), the genome-wide expression pattern of cancer and normal tissues from 27 patients were analyzed by a microarray-based method. Using an integrative systematic bioinformatics approach, we classified the differentially expressed genes in EGC. Interestingly, the more highly expressed genes in EGC exhibited the most significant correlation with cell migration and metastasis. This implies that, even at the early stage of gastric cancer, the molecular properties usually observed in late-stage cancer are already present. Furthermore, we have found a novel association between the expression pattern and molecular pathways of EGC and estrogen receptor α (ERα)-negative breast cancer through cross-experimental analysis. These results provide new insights into the biological properties of EGC, as well as yielding useful basic data for the study of molecular mechanisms of EGC carcinogenesis. Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide. Its prevalence is particularly high in East Asia, including countries such as China, Japan and Korea . The proTo address this, several microarray analyses in GC have been performed and have identified gene expression patterns that may be useful in the prognosis and diagnosis of the cancer ,6; howevHere, we focus on molecular understanding of EGC-specific expression patterns gained by employing a systematic approach, including function and pathway, as well as cross-experiment analyses of 27 pairs of EGC tissues and their normal counterparts. Interestingly, the function and pathway analyses show that the upregulated genes in EGC tissues correlate with cell migration and metastasis, events typical of late-stage cancer. In addition, we propose a novel association between EGC and estrogen receptor α (ERα)-negative breast cancer that was indicated by cross-experiment analysis, and which enables us to identify various associated phenotypes.Tissue samples were prospectively collected from patients who underwent gastric surgery or gastroscopy at the National Cancer Center (NCC) Hospital between 2008 and 2009. All tissues were obtained according to the protocols approved by the Institutional Review Board, NCC for the human subject guideline of NCC (NCCNCS-08-127) that is in accordance with the principles of the Declaration of Helsinki. The samples were obtained by endoscopic biopsies from gastric cancer patients who gave informed consent to the protocol. The samples were stored at –80°C. The clinical and pathological features of the patients are listed in Total RNA was extracted from gastric cancer and adjacent normal tissues from EGC patients using TRIzol reagent , followed by purification of the RNA using Qiagen RNeasy mini kit columns  according to the manufacturer’s instructions. RNA quality was evaluated using the Agilent 2100 Bioanalyzer  and concentration measured by Nanodrop 1000 . Only RNAs showing distinct 18S/28S ribosomal peak ratios of 1.5–2.0 in the Bioanalyzer (Agilent Technologies) and 260/280 ratios of 1.8–2.1 in the Nanodrop (Thermo Scientific) analyses were accepted for further analysis.www.bioconductor.org) ‘oligo’ package. The differentially expressed genes between EGC tissues and adjacent non-cancerous gastric tissues  were filtered by a fold-change cut-off of 1.5 and a P-value cut-off of 0.05.Genome-wide gene expression was analyzed in the 27-paired EGC tissue samples using Affymetrix GeneChip Human Exon1.0 ST Array . Target preparation and microarray processing procedures were carried out as described in the manufacturer’s instructions, and raw data were deposited in the NCBI Gene Expression Omnibus (GSE30727). The data were preprocessed by a default robust multi-array average (RMA) method implemented in the Bioconductor  was used to summarize the over-representation of the KEGG pathways (http://depts.washington.edu/l2l/) (We downloaded a Gene Ontology (GO) annotation file (gene_association.goa_human) and an ontology file (gene_ontology_ext.obo) from tutorial . In the pathways . The gendu/l2l/) .Two micrograms of total RNA were reverse transcribed with Superscript III reverse transcriptase (Invitrogen). Reverse transcription PCR (RT-PCR) was performed using 5 ng cDNA for 1 cycle at 94°C for 2 min, followed by 32–35 cycles of 94°C for 20 sec, 60°C for 40 sec and 72°C for 30 sec, using gene-specific primers . Gene exwww.ncbi.nlm.nih.gov/geo) and EBI ArrayExpress (www.ebi.ac.uk/arrayexpress): GSE19536 for ERα-negative breast cancers  from the 27 pairs of EGC tissue and their adjacent normal tissue. The P-value cut-off of 0.05 in t-tests, and the fold-change cut-off of 1.5 or 1/1.5 for up- and downregulated genes, respectively, was used for selection. We identified 556 upregulated genes and 417 downregulated genes. The differentially expressed genes were then fed into function, pathway and cross-experiment analyses to acquire a deeper understanding of the molecular basis of EGC.http://www.cytoscape.org/) was used to explore the molecular function and biological processes in GO.The BiNGO plug-in on the Cytoscape platform  . Moreovehttp://david.abcc.ncifcrf.gov/) was used to inspect the KEGG biological pathways associated with the differently expressed genes in EGC.The DAVID tool , which are well known cell migration-related genes. MMPs are also known to play critical roles in the regulation of cell invasion by ECM proteolysis, as well as by processing cytokine precursors in the chemokine network.We analyzed the expression pattern of 7 MMPs  within the upregulated gene data in EGC tissues. As expected, and consistent with the microarray data where MMPs were upregulated 1.56- to 8.68-fold , RT-PCR In order to investigate similar molecular signatures between EGC and other cancer types, we compared our data of differentially expressed genes with a public gene expression signature warehouse, L2L. This revealed that the upregulated genes in EGC most significantly correlated with the gene expression signature of ERα-negative breast cancer . As summThe same L2L analysis was applied to the downregulated genes in EGC . InteresTo validate the result of the L2L analysis showing a relationship between EGC and ERα-negative breast cancer, we performed a hierarchical clustering analysis. The expression datasets of the differently expressed genes in EGC (556 upregulated gene symbols and 417 downregulated gene symbols), ERα-negative breast cancer and 4 additional cancers  were used in an unsupervised hierarchical clustering analysis. As in the L2L analysis, the results indicated that EGC correlated most closely with the ERα-negative cancer than with the other 4 cancers .When we inspected the expression levels (z-scores) of the 7 MMP genes , the resWe analyzed the microarray data generated from pairs of tumor tissue and their adjacent non-cancerous tissue, obtained from 27 EGC patients. The gene expression data were subjected to functional and pathway analyses, as well as gene expression signature comparison . This led to 2 novel findings: i) the functional and pathway analyses suggested that metastasis-related biological processes may already be highly expressed even in the early stage of gastric cancer, and ii) the gene expression pattern of EGC is closely aligned to that of ERα-negative breast cancer.et al suggested a carcinogenesis model  signaling that induces Cox-2, followed by activation of cell proliferation (feration . In factferation , Fig. 5.In conclusion, we have analyzed the differentially expressed genes in EGC patients using an integrative systematic approach. We found that genes highly expressed in EGC are involved in cell migration- and metastasis-related functions typically observed in late-stage cancer. Also, EGC may be intrinsically similar to ERα-negative breast cancer, by sharing immune-related signaling events, which is further dissected in both cancer types. The functional roles of the downregulated genes in EGC carcinogenesis remain to be elucidated in future.


The good barrier properties are due to the compact and dense structure of the films, as revealed by field-emission scanning electron microscopy. A relationship between OTR and the structure of the corresponding nanofibril-based films was confirmed.Cellulose nanofibrils have been proposed for novel barrier concepts, based on their capability to form smooth, strong and transparent films, with high oxygen barrier properties. A series of cellulose-based films were manufactured and tested with respect to their oxygen transmission rate (OTR) capabilities. The obtained OTR levels were considerably better than the levels recommended for packaging applications. Part of the nanofibrillated material applied in this study was produced with 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) mediated oxidation as pretreatment. Films made of TEMPO-pretreated samples yielded lower OTR values. The minimum obtained OTR value was 3.0 mL m Cellulose nanofibrils are nano-components of a cellulosic material produced through a fibrillation process . SeveralThe density of nanofibril-based films is an important property to quantify, considering their potential application as barriers in packaging. The density gives an indication of the consolidation of the films during production. The more consolidated a given film is, the larger the density. Yang et al.  reportedThe purpose of this study is to shed light on the structure of nanofibril-based films and on how this complex structure limits the oxygen transmission rate through the material. A relationship between the structure of a series of nanofibril-based films and the corresponding OTR levels is confirmed.Eucalyptus and Pinus radiata, as described by Syverud et al. [A series of nanofibril qualities were utilized in this study. The nanofibril qualities were produced from d et al. . Some ofd et al. . 2,2,6,6d et al. .The kraft pulp fibres (0.5% consistency) were homogenized with a Rannie 15 type 12.56X homogenizer , operated at 1,000 bar pressure. The fibrillated materials were collected after three and five passes through the homogenizer (Table Films (F01 to F08) were prepared in plastic petri dishes by free drying. The drying temperature was 23°C. Films of samples F01, F02 and F07 were additionally made in a cylindrical mould . The filδf) are given by the relationship γ/τf, where γ and τf correspond to the grammage and mean thickness of the films, respectively.A scanning electron microscopy (SEM) cross-sectional analysis (thickness and roughness) was performed in backscatter electron imaging (BEI) mode as described by Chinga-Carrasco et al. . The app2, with a lateral resolution of 1 μm. The AFM analysis was performed on local areas of 5 × 5 μm2, with a lateral resolution of 10 nm.A surface structural quantification based on laser profilometry  and atomic force microscopy  was performed on the films, as described by Chinga-Carrasco et al.  and SyveThe embedded films were exposed to a small quantity of water to induce a delamination of the cross-sectional structure. After drying, the samples were then covered with a conductive layer. Images were acquired at various magnifications with a field-emission SEM . The images were acquired in secondary electron imaging (SEM-SEI) mode. In addition, a field-emission SEM analysis, applying the inlens detector, was performed for revealing the surface nanostructure of the films.® 1/50 test system  at 50% relative humidity and 23°C.The OTR was measured with a Mocon OX-TRANz-direction of the films  [-2 day-1 atm-1) [OTR values lower than 20 mL mications . The OTR1 atm-1)  and cell1 atm-1) . For a c1 atm-1) .-2 day-1 atm-1. This seems to confirm the poor connectivity of the pores in the film structures.It is worth to notice that despite the major differences between the assessed films with respect to the nanofibril morphology and pore structure Figure , the OTRFor a given grammage, the more porous a structure is, the thicker the corresponding film. OTR values can thus be related to the corresponding film thicknesses, as exemplified in Figure Considering the positive correlations between a given film structure and the corresponding OTR levels suggests that increasing the density decreases the OTR. This is confirmed in Figure Eucalyptus nanofibril-based films, made by free drying, yield lower densities than the corresponding P. radiata nanofibril-based films. One thus expected higher OTR values for the Eucalyptus nanofibril-based films. Eucalyptus films yield, however, similar OTR values at lower density (apparent higher porosity) compared to P. radiata . The density-OTR correlations should probably be translated to higher density levels, approaching the OTR-density correlation of the P. radiata nanofibril-based films. Films made under restrained conditions were analysed to verify this assumption. The OTR-density values of the films made under restrained conditions yielded variable results, probably due to the uneven structure caused by the formation procedure. However, the OTR-density measurements are in the same range as the measurements of the free-dried films and follow the same trend, i.e. increasing density reduces the OTR. This is a confirmation of the expected OTR-density relationship.The d et al. . Shrinka2) were considerably better than the levels recommended for packaging applications. Films made of TEMPO-pretreated samples yielded lower OTR values. The minimum obtained OTR was 3.0 mL m-2 day-1 atm-1 with a corresponding oxygen permeability of 0.04 mL mm m-2 day-1 atm-1, tested at 50% relative humidity. The good barrier properties are due to the compact and dense structure of the films. A relationship between OTR and the structure of the corresponding nanofibril-based films was confirmed.The obtained OTR levels of the films used in this study (grammage 20 g/mAFM: atomic force microscopy; BEI: backscatter electron imaging; LP: laser profilometry; OTR: oxygen transmission rate; SEI: secondary electron imaging; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TEMPO: 2,2,6,6-tetramethylpiperidinyl-1-oxyl.The authors declare that they have no competing interests.GC-C was involved in the production and characterisation of cellulose nanofibrils; performed the LP, SEM and FESEM analyses; wrote the manuscript and performed the corresponding revisions. KS was involved in the production and characterisation of cellulose nanofibrils, was responsible for the oxygen transmission rate measurement and, has been involved in revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.


E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200–300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.Bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in  E. coli to ethanol. Models for evolving regulatory networks were developed by Crombach et al. [Since the long term evolution experiment of Lenski et al. , laborat et al.  and Xie  et al. . Constra et al. .Here, we present the concept of an adaptive evolution experiment in a bioreactor. In such a process, the evolutive pressure on the microorganisms for either fast growth or optimal biomass yield on a limiting substrate can be used to attain or improve the production of a desired compound. Motivated to know possible endpoints of the evolution experiment, we developed an algorithm for computing the endpoints of such an experiment. These endpoints are alternative flux distributions for the circumvention of a metabolic block. We further examined the role of a regulatory network in the usage of the alternative flux distribution and we validated the model by microarray analysis.Escherichia coli which lacks both the pyruvate kinases PykA/PykF and the phosphoenolpyruvate carboxylase (Ppc). The pyruvate kinases are expected to be the main source of pyruvate on a glycerol minimal medium [pyc) of Corynebacterium glutamicum was inserted into the chromosomal malEG locus under control of the tac-promotor. The Pyc enzyme catalyzes the carboxylation of pyruvate to form oxaloacetate [The experiment utilized a mutant of the intestinal bacterium  medium . The Ppc medium . As replacetate .malE::pyc is pyruvate-auxotrophic . The stoichiometric information for balanced compounds was described by the matrix n0 and ne as the number of balanced and unbalanced compounds, respectively. To denote the external substrate availability, the vector bglyc was negative and if lactate was not available blac=0). Furthermore, the growth rate was fixed to the dilution rate Jμ = D due to chemostat conditions. Since thermodynamic constraints on reactions exist, some reactions are irreversible and the direction of the flux is fixed. The following equality and inequality constraints were collected in the constraint set The metabolic compounds which can be further analyzed by using objective functions for optimization.f = cτJ. Minimizing f  results in an optimal value fopt and a particular solution Jopt. Applying objective functions will often result in non-unique optima. Consequently, the set of optimal solutions has to be further analyzed. By extending the constraints in Equation (1) with the equation fopt = cτJ enforcing the optimal objective function value, a new constraint set Properties of the constraint set Yield: The yield is defined as growth per substrate uptake μ is determined by the dilution rate D. For optimization purposes, the yield can be maximized by minimizing the substrate uptake JSup,→ min.Turnover rate: With a balanced metabolite the consumption and production rate are equal, which is a measure for the turnover. We define the turnover rate as the production rate of a compound. The objective function Ci with νi,jas a stoichiometric coefficient. A yield optimal minimal turnover rate (YMTR) was computed by extending the constraints with the fixed minimal substrate uptake rate results in a minimal turnover rate (MTR) of a balanced compound Ciwas developed   whether 2. Construct a constraint set l be the number of constraints in 3. Starting with k = 1, compute the optimal yields of k reactions. All 4. If there is a feasible solution for the set restricting all previously found solutions k and repeat step 3.Ci were obtained. To reconstruct the alternative pathways from a substrate to the MOI, this algorithm had to be used recursively. At first it was applied to the MOI, then to the reactants of the last reactions to the MOI and then to the reactants of those reactions and so on.In this manner, all minimal combinations of alternative reactions to produce The computational effort is high due to the recursive usage of the algorithm. Because the point of interest was the buildup of a metabolite’s carbon core, it was only necessary to track the reactants that carry parts of the carbon core for the metabolite. To decide whether or not a reactant contributes to the carbon core, we used the following equivalence relation:Given a set of cofactorsCo, two metabolitesCi and Cjare equivalent up toCo, the regulatory network is not necessarily optimal any more. Consequently, random mutations leading to an altered regulatory network were expected by Crombach et al.  as a driWe intended to study how transcriptional regulation affects the availability of enzymes that are essential for the predicted alternative pathways Table . Therefoσand anti-σfactor knockouts. Only 41 of the evaluated factors are included in the iMC1010v2 model. The environmental conditions of the experiments were expressed in a constraint set such as σ factors changed the TFN and resulted in a changed constraint set k denoted the factor which was deleted. The objective function of minimal substrate uptake was evaluated on the constraint sets malE::pyc.We used data of the transcription factor knockout study of Haverkorn et al.  in ordermalE::pyc and the evolved strains showed different growth features. In fact eight alternative metabolic pathways exist to circumvent the metabolic block of regular pyruvate formation. This raised the question why F41malE::pyc was not able to use these eight alternative metabolic pathways on minimal media (MM) with glycerol. After a cultivation phase of F41malE::pyc in MM with glycerol, growth could be obtained by adding lactate. This indicated that F41malE::pyc was not poisoned by toxic metabolites. The useability of some of the eight alternative pathways was proven by the sheer existence of the evolved pyruvate prototrophic strains. Therefore, we examined the hypothesis if transcriptional regulation prevents the transcription of genes which are essential for the usage of the alternative pathways. We checked how the transcriptional network impacts the eight alternative pathways  , the strThe results indicated that estimating from the microarray data, the enzymatic capability of using the alternative pathways was available in all three compared strains. The TFN did not predict a transcriptional downregulation of the pathways in most cases, which was in accordance with the microarray data, except for the Entner-Doudoroff-path and alternative acetyl-CoA synthesis path. This means that the hypothesis of restricting the alternative pathways by transcriptional regulation did not hold.malE::pyc on glycerol with an additional pyruvate source. The introduced algorithm for the computation of alternative pathways was employed to predict pathways from glycerol to pyruvate as possible endpoints of evolution for the strain F41malE::pyc. The evolution experiment with F41malE::pyc resulted in five evolved strains. This proved that the usage of alternative pathways was possible after adaptive evolution. However, F41malE::pyc was unable to grow without a pyruvate source. We assumed that transcriptional restriction of the predicted pathways hindered the growth. Therefore, a Boolean transcription factor network (TFN) was employed to further restrict the solutions of the metabolic network (MN). The prediction of the TFN together with microarray analysis revealed that in this case it is improbable that transcriptional regulation was exclusively responsible for the activation of the proposed alternative pathways during adaptation. It was shown that mRNA of genes which are important for the functionality of the predicted pathways were present in all compared strains.We have illustrated the idea of an adaptive evolution experiment in a chemostat bioreactor, where mutation and selection led to circumvention of a metabolic block. Constraint-based methods were utilized to identify targets for blocks and to predict alternative pathways for this circumvention. We performed the experiment with a pyruvate-auxotrophic strain F41However, a clear elucidation of the course of genetic events during adaptation was not yet possible. Metabolic feedbacks and non-regulatory effects may play an important role. We believe that the TFN will help to support further analysis by giving the possibility to determine the regulatory effects of metabolic and environmental signals and to distinguish between cause and effect of the up/down regulation of a gene. This will warrant further study in the field of transcription factor networks and their input in order to understand the whole sequence of events during adaptive evolution.2PO4·2 H2O, 11.5 g K2PO4, 2.64 g (NH4)2SO4, 0.74 g MgSO4·7H2O, 14.7 mg CaCl2·2H2O, 13.5 mg ZnCl2, 2.8 mg FeSO4·7H2O, 10μl 1N HCl, 20 mg Thiamine, 0.2 mM IPTG per liter. In shaking flask experiments 0.5% (w/v) glycerol was used as carbon source. When a mixture of glycerol and D,L-lactate was used the concentrations were 0.4% glycerol and 0.1% DL-lactate in order to have constant carbon availability in all experiments. Cells were cultivated in 250 ml shaking flasks filled with 25 ml growth medium. Prior to use, the cells were streaked onto LB-agar plates freshly from −80°C frozen stocks and incubated overnight at 37°C. Single colonies from LB-agarplates were then adapted to growth on minimal medium on MM agar plates for three days. Cultures were initiated directly from MM agar plates at OD600 = 0.1. After overnight incubation at 37°C without shaking the cells were grown at 37°C and 70 rpm.The strains used in the experiments are listed in Table 2 was kept above 50%. The feeding contained MM with 5 g/L glycerol and lactate concentrations in the range from 0.125 g/L down to 0 g/L of 95% L-lactate. The last 100 fermentation hours the feeding contained no lactate. The glycerol concentration measured in the fermentation broth was close to zero.Chemostat fermentations were performed in a Bioengineering fermentor KLF at 37°C, with stirring rate of 500–1000 rpm, an input air of 1 L/min, controlled pH at 7 and pOFor genome resequencing the cells were grown to stationary phase. Chromosomal DNA was isolated via Phenol/Chloroform precipitation . The resmalE::pyc were compared after growth on minimal medium containing glycerol and D,L-lactate as carbon source. Cells were harvested after reaching OD600 = 0.8. The DNA chips were custom-synthesized by Agilent company and processed according to the manufacture’s instruction. A complete description of transcript data will be published elsewhere, but can be obtained from the authors directly.For transcriptome analysis strains K98-62 and LJ110 were compared after growth on minimal medium containing glycerol as carbon source and K98-62 and F412 spot intensities over all biological replicates and colors. If the average expression value of a mRNA was measured below 6 units, it is uncertain that the mRNA was present in the probe. If the fold change value was not significantly different from zero and the average expression value was above 7.0, we assumed that mRNA of a gene was present in both compared strains. Observing significant fold change values, we studied the strain specific average expression to assess whether mRNA was present or not. The mean average expression value over all spots was 6.93 in the comparison K98-62 versus wild type and 7.37 for K98-62 versus F41malE::pyc. Data to estimate the enzymatic capability of the predicted pathways was included in Additional file The average expression is the mean value of all normalized LogJATPm in JATPm = 8.39[mmolh−1gDCW−1 from Feist et al. [In Equation (1), we regarded also ATP requirements for the maintenance metabolism  et al. , which iCombination of iAF1260 and iMC1010v2: The computation of a regulatory model combined with metabolic model was outlined by Covert et al. . The netaccoa: Acetyl-CoA; prpp: 5-Phospho-α-D-ribose-1-diphosphate; acgam6p: N-Acetyl-Dglucosamine6phosphate; pser: Phospho-L-serine; akg: Oxoglutarate; pyr: Pyruvate; anth: Anthranilate; r5p: α-D-Ribose-5-phosphate; aspsa: L-Aspartate-4-semialdehyde; ser-D/L: D/L-Serine; chor: Chorismate; skm: Shikimate; cys-L: L-Cysteine; succ: Succinate; dha: Dihydroxyacetone; thf: Tetrahydrofolate; dhap: Dihydroxyacetone-phosphate; thr-L: L-Threonine; e4p: D-Erythrose-4-phosphate; trp-L: L-Tryptophan; fdp: D-Fructose-1-6-bisphosphate; uacgam: UDP-N-Acetyl-D-glucosamine; for: Formate; 10fthf: 10-Formyltetrahydrofolate; fum: Fumarate; 13dpg: 3-Phospho-D-glyceroyl-phosphate; f6p: D-Fructose-6-phosphate; 2ddg6p: 2-Dehydro3deoxy-D-gluconate6phosphate; glyc: Glycerol; 2dr1p: 2-Deoxy-D-ribose-1-phosphate; glyc-R: R-Glycerate; 2mcit: 2-Methylcitrate; glyc3p: Glycerol-3-phosphate; 2obut: 2-Oxobutanoate; g3p: Glyceraldehyde-3-phosphate; 2pg: D-Glycerate-2-phosphate; ichor: Isochorismate; 23ddhb: 2-3-Dihydro-2-3-dihydroxybenzoate; lac-D/L: D/L-Lactate; 3pg: 3-Phospho-D-glycerate; malACP: Malonyl-acyl-carrier-protein; 3php: 3-Phosphohydroxypyruvate; malcoa: Malonyl-CoA; 4abz: 4-Aminobenzoate; mlthf: 5-10-Methylenetetrahydrofolate; 4adcho: 4-Amino-4-deoxychorismate; mthgxl: Methylglyoxal; 4hbz: 4-Hydroxybenzoate; oaa: Oxaloacetate; 4pasp: 4-Phospho-L-aspartate; 6pgc: 6-Phospho-D-gluconate.The authors declare that they have no competing interests.RF has written the manuscript, has done modeling and experimental study and did conceptual study. KG has done experimental study including transcript analysis. GV has done the computations for the model analysis of transcription factor knockout study. JK and SS have supervised the study of GV and did theoretical studies with the transcriptional network. MB and OS contributed to the design of the project. GS contributed to the design of the project and to the interpretation of data. ME contributed to the design of the project, has developed a preliminary version of the computational algorithm, has done conceptual study on the article and helped to interpret the data. All authors read and approved the final manuscript.Alternative pathway classes. Additional file 1 contains detailed information about the alternative pathway classes from Section “Abbreviations”.click here for fileModifications of iMC1010v2, input signals and predicted inactive genes. Additional file 2 describes how iMC1010v2 can be adapted to the metabolic model iAF1260. It contains input signals for three environmental conditions and the according outputs.click here for file